Analytical Cascades of Enzymes for Sensitive Detection of Structural Variations in Protein Samples

Abstract

Protein function critically depends on structure. However, current analytical tools to monitor consistent higher-order structure with high sensitivity, as for instance required in the development of biopharmaceuticals, are limited. To complement existing assays, we present the analytical cascade of enzymes (ACE), a method based on enzymatic modifications of target proteins, which serve to exponentially amplify structural differences between them. The method enables conformational and chemical fingerprinting of closely related proteins, allowing for the sensitive detection of heterogeneities in protein preparations with high precision. Using this method, we detect protein variants differing in conformation only, as well as structural changes induced by diverse covalent modifications. Additionally, we employ this method to identify the nature of structural variants. Moreover, the ACE method should help to address the limited reproducibility in fundamental research, which partly relates to sample heterogeneities.

The nanotopography of SiO2particles impacts the selectivity and 3D fold of bound allergens

“…To determine the structural alterations upon particle binding, the two-step analytical cascade of enzymes (ACE), as previously described by Hollerweger et al , 31 was performed. This method uses the sequential application of enzymes to amplify the signal of small conformational changes of a protein and makes them detectable using simple laboratory methods.…”

“…28,[58][59][60] Nanotopography impacts the 3D fold of NP-bound allergen Next, we determined the conformational changes of Bet v 1 following its binding to the two different types of SiO 2 NPs. Circular dichroism (CD) spectroscopy and a two-step analytical cascade of enzymes (ACE) 31 were used for the detection of secondary structural changes and their impact on the 3D fold of Bet v 1 (Fig. 3a).…”

“…Moreover, to analyse the influence of particle binding on the 3D fold of proteins, circular dichroism (CD) spectroscopy and a novel enzymatic assay, termed the analytical cascade of enzymes (ACE), were used. 31 To investigate whether protein-NP binding favours a specific orientation of the bound allergen at the particle surface, limited proteolysis approaches with different proteases were employed. Finally, to uncover modified biological responses such as potential changes in the availability of the allergenic epitopes, direct ELISA and basophil degranulation assays were performed on the particle-bound vs. free allergen after incubation with either the allergen–NP conjugates or the unbound allergen.…”

The Fluorescent Enzyme Cascade Detects Low Abundance Protein Modifications Suitable for the Assembly of Functionally Annotated Modificatome Databases

“…[16][17] We recently presented a novel method to address these issues, the analytical cascade of enzymes. [18] The initial, chemiluminescent version of the enzyme cascade (referred to as ACE in the original publication and cEC from here on) allows to detect small variations between protein samples, such as folding variations. To this end, the protein samples of interest are treated with a series of enzymes in order to introduce analytical modifications that exponentially enhance structural variations reflecting heterogeneities in protein samples (cf.…”

“…We recently presented a novel method to address these issues, the analytical cascade of enzymes [18] . The initial, chemiluminescent version of the enzyme cascade (referred to as ACE in the original publication and cEC from here on) allows to detect small variations between protein samples, such as folding variations.…”